Báo cáo khoa học: "Reinvigoration treatments for the micropropagation of mature chestnut trees" pptx

Original articleReinvigoration treatments for the micropropagation MC Sánchez A Ballester, AM Vieitez Instituto de Investigaciones Agrobiológicas de Calicia CSIC, Apartado 122, 15080 San

Original article

Reinvigoration treatments for the micropropagation

MC Sánchez A Ballester, AM Vieitez

Instituto de Investigaciones Agrobiológicas de Calicia (CSIC),

Apartado 122, 15080 Santiago de Compostela, Spain

(Received 20 November 1995; accepted 11 June 1996)

Summary - Crown material from five adult chestnut trees was given different reinvigoration

treat-ments, such as 6-benzylaminopurine (BA) applications (spray or pulse) to forced cuttings, and juve-nile grafting alone or combined with BA sprays, then used for the establishment in vitro The in vitro performance, in terms of establishment, multiplication and rooting, of both untreated and treated material was compared Grafting alone or in combination with BA spray greatly increased the in vitro reactivity of crown-derived explants By combining in vivo pretreatments and a horizontal reculturing system, crown-derived microshoots exhibited maximum rooting rates, similar to those found for cultures from basal shoots of the same tree in previous work.

chestnut / horizontal reculture / micropropagation / mature trees / partial rejuvenation

Résumé - Traitements de rajeunissement de châtaigniers adultes Pour faciliter l’établissement

in vitro du châtaignier, différents traitements, tels que des applications de 6-benzylaminopurine (BA,

pulvérisation ou trempage de 2 h), le greffage sur porte-greffe juvénile, seul ou combiné avec des pul-vérisations de BA, ont été appliqués au matériel prélevé dans la couronne de cinq châtaigniers adultes On a comparé le comportement durant l’établissement, la multiplication et l’enracinement in vitro, du matériel non traité (témoin) et du matériel rajeuni Durant l’établissement in vitro, la réac-tivité du matériel témoin a été relativement faible pour tous les clones (variations entre 0 et 22 %) Le greffage, seul ou combiné avec des pulvérisations de BA, augmente significativement la réactivité des

explants provenant de la cime des arbres, atteignant 94 % pour le clone HV En ce qui concerne la phase

de multiplication, les meilleurs résultats ont été obtenus avec les microboutures dérivées du matériel provenant de la couronne, pulvérisé avec BA (C+S), ainsi qu’avec ceux des greffes pulvérisées avec

BA (G+S) Dans les deux cas, on a obtenu des valeurs bien supérieures à celles du témoin Un

com-portement similaire a été observé dans leur aptitude à l’enracinement Ces résultats montrent que les traitements C+S et G+S induisent un certain rajeunissement du matériel adulte En combinant les prétraitements in vivo avec un système de culture répétée des explants in vitro en position horizon-tale (recyclage), les microboutures dérivées de la couronne présentent un taux de multiplication

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significativement supérieur explants position recyclés.

De plus, avec ce système on a obtenu des taux d’enracinement (63 et 28 % pour les clones 431 et HV, respectivement) similaires à ceux précédemment trouvés dans des cultures dérivées de rejets de la base

du même arbre.

arbres adultes / châtaignier / micropropagation / reculture horizontale / rajeunissement partiel

INTRODUCTION

One of the limiting factors for the

micro-propagation of recalcitrant mature trees is

the loss of morphogenetic capacity of

explants as the tree ages In vitro cloning of

mature chestnut trees has been successfully

accomplished from material retaining

phys-iologically juvenile characteristics, such as

basal shoots and stump sprouts (Biondi et

al, 1981; Vieitez et al, 1983; Chauvin and

Salesses, 1988) In contrast, it is well known

that the micropropagation of material from

the crown of mature trees still remains very

difficult In a previous paper, we reported

for five chestnut clones the morphogenetic

capacity greater for basal shoot-derived

explants than for crown branch-derived

ones Shoot cultures were established from

both sources, but the in vitro performance of

crown explants, in terms of reactivity,

mul-tiplication and rooting rates was very poor

(Sánchez and Vieitez, 1991) For this reason,

the success in micropropagating a selected

chestnut tree is, to a large extent, a function

of the availability of juvenile or

reinvigo-rated material

Although mature phenotypic

character-istics are stably maintained and transmitted

once maturity has been attained, they are

reversible under certain conditions (Hackett,

1985) Therefore, as the morphogenetic

response of explants in vitro is greatly

influ-enced by their maturation state (Hackett and

Murray, 1993), it is interesting to obtain

reinvigorated or partially rejuvenated

mate-rial from a desirable mature tree by several

experimental approaches According to

Pierik ( 1990), a majority of the treatments

used cause an increase in vigor and rooting

(reinvigoration), with true rejuvenation being

difficult to achieve The rooting capacity of

mature material was improved in different

woody species by treatments including

severe pruning (Howard et al, 1989), serial

rooting of cuttings (Morgan et al, 1980),

juvenile grafting (Franclet, 1981; Ballester

et al, 1990), spraying with cytokinins

(Bouri-quet et al, 1985), partial etiolation (Ballester

et al, 1989) as well as by inducing epicormic

shoots in crown branches of mature trees

(Vieitez et al, 1994) or stem sections (Evers

et al, 1993) In vitro rejuvenation methods,

such as meristem culture, serial

micrograft-ing and reculture of the same original explant, among others, have also been

suc-cessful

In this study, different reinvigoration

pre-treatments were applied to crown material of five chestnut clones and the

micropropaga-tion ability (in vitro establishment,

multi-plication and rooting) was compared to

untreated controls In addition, an in vitro

rejuvenation method was assayed in the established cultures

Crown cuttings of five mature chestnut trees, referred to as HV, 431, A2, A3 and P 1 were col-lected during the rest period (December-Jan-uary) and stored at 4 °C for 3 months, before being forced to flush or grafted The HV and 431

trees, aged 30 and 15 years, respectively, are

Castanea sativa Mill x C crenata Siebeld and Zuce hybrids resistant to Phytophtora cambivora

and P cinnamomi The other three clones were

obtained from natural stands of healthy C sativa Mill trees aged 50 (A2), 40 (A3) and 80 (P1)

A diagram of the material and different

tures is shown in figure 1.

Spray treatment

In March the cuttings from all five trees,

col-lected the previous December, were separated

for each clone in two different sets, placed in

water and forced to flush in a growth cabinet.

During the flushing period, one set of cuttings

of each clone was sprayed with a sterilized 222

μM solution of 6-benzylaminopurine (BA) three

times a week, and the other set was used as a

control After 2 weeks, the new shoots that

devel-oped from both untreated and BA treated

cut-tings were collected, and used as the source of

ini-tial explants (C and C+S) These shoots were

sterilized, subdivided into 5 mm shoot tips and

nodes bearing one or two axillary buds and

estab-lished in vitro.

Pulse treatment

To stimulate the in vitro response of crown

explants, a subset of the sterilized shoot tips and nodes derived from unsprayed cuttings of HV

and 431 trees were placed for 2 h in Petri dish plates (eight explants per plate) containing a fil-ter-sterilized solution of 111 μM of BA After this pulse treatment, explants (C+P) were

trans-ferred to in vitro culture conditions.

Juvenile grafting

In April, scions (3-4 cm long) bearing two to

three buds were taken from stored January

cut-tings of HV, A3 and 431 trees and grafted onto 2-week-old seedlings, which were obtained by ger-minating seeds of the HV tree Grafting was

performed as per Vieitez and Vieitez (1981) by

removing seedling epicotyls inserting

the scion into the split hypocotyl The grafts were

kept for 5 weeks in a growth chamber to force

flushing of lateral buds Three weeks later, some

grafts were sprayed with 222 μM BA solution

three times a week for 2 weeks Newly grown

shoots from unsprayed and sprayed grafts were

used as the source of initial in vitro explants (G

and G+S)

In vitro culture

Micropropagation procedures were carried out

as described elsewhere (Vieitez et al, 1983;

Sánchez and Vieitez, 1991) Briefly, all shoots

used for initial explants were stripped of leaves

and surface-sterilized by successive immersion

for 30 s in ethanol and 10 min in 12%

commer-cial bleach (40 g·Lof active chlorine), followed

by three rinses in sterile distilled water

Steril-ized explants from all sources were placed

ver-tically in 20 x 150 mm test tubes containing 15

mL of establishment medium which consisted

of Gresshoff and Doy’s medium (1972)

supple-mented with 2.22 μM BA, 30 g·L sucrose and

6 g·L Difco agar The pH of the medium was

adjusted to 5.5-5.6 before autoclaving at 121 °C

for 20 min One day after implantation in vitro,

the explants were moved to a different place

within the same tube to reduce the negative effect

of the blackening of the medium probably due

to phenol oxidation and exudation All explants

were then transferred to a fresh medium every

2 weeks to overcome the negative effect of this

exudation After 6 weeks, the newly developed

shoots, cut into 8 mm long shoot tips and nodal

segments, were subcultured vertically on a fresh

medium with 0.89 μM BA (multiplication

medium) to start the shoot multiplication stage

Unless otherwise stated, subculturing was

car-ried out every month except for HV explants,

which were transferred, after 3 weeks on BA,

for 2 more weeks on a medium containing 0.46

μM of zeatin, giving a 5 week multiplication

cycle.

For rooting, the base of the shoots was dipped

in 4.9 mM indole-3-butyric acid (IBA) solution

for 2-3 min, and transferred to fresh medium

without BA and with macronutrients reduced to

one-third strength (rooting medium) Shoot

mul-tiplication and rooting experiments were carried

out after subculturing for at least 1 year.

grown under

orescent lamps delivering 30 μmol m s-1 dur-ing a 16 h photoperiod, with day/night

tempera-tures of 24/20 °C.

Recycling of the same horizontal

explant

The influence of repeated culture of the same

explant (recycling) placed horizontally on the media on multiplication rates and rooting ability

of shoots obtained from multiplication cultures

was investigated on clones HV and 431 Microshoots (20-25 mm long) were harvested from shoot proliferating cultures which were ini-tiated from C+S explants for clone 431 or G+S explants in the case of clone HV Shoots were

decapitated and placed horizontally in glass jars (six per jar) containing 70 mL of multiplication medium At the end of 4 (clone 431) or 5 weeks (clone HV) all the new axillary bud-derived shoots were harvested and used for multiplication

or rooting experiments Once the shoots were

harvested (first cycle), the original explant was

recycled on a fresh medium (second cycle) In

successive recultures, the shoots were developed from axillary buds that failed to elongate in the original explant or from the axillary buds located

in the stump of previously harvested shoots Each original explant was recycled four times in the

case of clone HV and up to eight times for clone

431 The reculture period of the clone 431 was

reduced to 2 weeks in the third and successive recultures due to the fast shoot growth, while a 5 week period was maintained in clone HV Root-ing capacity was evaluated in shoots taken from successive reculture cycles.

Data collection and statistical analysis

At the end of the establishment stage (initial cul-tures), the in vitro reactivity (defined as the per-centage of surviving explants with shoot devel-opment), the number of shoots greater than 8

mm per responsive explant, and the length of the tallest shoot per explant were recorded In shoot

multiplication experiments, the shoot mean

num-ber, the tallest shoot as well as the number of 8

mm nodal and shoot tip segments produced per explant were evaluated In rooting experiments, the of rooted shoots, the number of

per rooted shoot and the longest length

for each rooted shoot were assessed.

In in vitro establishment experiments, the

number of initial explants is shown in table I.

For each clone and treatment, 18 replicates were

used in shoot multiplication and rooting

experi-ments, and the experiments were repeated four

times The multiplication data and the rooting

percentages of recycling experiments were

ana-lyzed by one-way analysis of variance (ANOVA)

followed by least significant difference (LSD)

test at P < 0.05 to compare means Arcsine

trans-formation was applied to rooting percentage data

prior to analysis Non-transformed data are

pre-sent in the tables and figures The significance

of differences among reactivity percentages (table

I) and among rooting percentages (table II) were

analyzed by the test of independency (G-test)

(Sokal and Rolf, 1981).

In all five clones there were no differences

in the sprouting capacity between BA

sprayed and untreated crown cuttings forced

to flush in the growth cabinet Most buds

just swelled and remained green for about 1

week, then became brown or necrotic; how-ever, 10-35%, depending on the clone, broke and grew to a maximum size of 1 cm, but failed to elongate further BA treatment

did not improve the bud breaking of crown

cuttings forced to flush In contrast, when

grafting was carried out, vigorous shoots,

20-25 cm long, were obtained and used as

the source of G and G+S explants (fig 2).

The success of grafts performed with scions

of clones A3, 431 and HV was 30, 43 and

55%, respectively.

The effect of the different treatments

applied to the starting material on in vitro

establishment is shown in table I

Contam-ination rates did not exceed 15%, with the

highest values for explants taken from

mate-rial that had been sprayed with a BA solution

(C+S and G+S) Within each clone, the

per-centage of surviving explants with shoot

development (reactivity) depended on the

treatment The in vitro response of C

explants was relatively low in all tested

clones, with A3 being the most responsive

clone (21.7%) Untreated HV explants were

totally unreactive The reactivity of C

explants (clones HV and 431) was not

improved by a BA pulse whereas for HV

C+S explants it was significantly increased

Moreover, percentage

explants was also increased (not

signifi-cantly) in C+S explants for the other four clones In grafted material (G explants), the

reactivity was significantly greater than in the controls (C explants) and was further increased with a BA spray (G+S explants).

For clone HV, G+S explants exhibited a

reactivity significantly higher than those observed from grafted plants Within each clone, the number of shoots per explant and the longest shoot length were not affected by

any treatment.

In shoot multiplication stage,

pretreat-ments of starting material had a greater

effect on the number of segments than on

the shoot number Pretreatments had no

effect on shoot numbers of A2 and A3

clones, whereas in clones P 1 and HV the

significant best values were found in C+S or

G+S cultures, respectively (data not shown). Shoot multiplication rates, in terms of mean

number of 8 mm nodal and shoot tip

seg-ments produced per explant is shown in table

II Generally, treatments including spray

application (C+S and G+S explants)

afforded, in a significant way, the best mean segment number, except in P1 and 431

cul-tures The length of the tallest shoot was not

affected by treatments, ranging from 10 to

20 mm depending on the genotype (data not

shown) The rooting capacity of microshoots derived from different pretreatments was

very low (table II) Significantly higher

root-ing values in comparison to controls were

obtained when either C+S (A2, A3 and 431 )

or G+S (HV and 431) shoots were used, whereas no effects on root number and

longest root length were observed

Figure 3 shows the effects of explant ori-entation and recycling on shoot

multiplica-tion of clone 431 The average number of shoots produced in explants cultured

hori-zontally once (H1) was significantly greater

than in vertically placed explants (V1)

Pro-duction of high numbers of shoots was

maintained by recycling the same

horizon-explant

system, the number of shoots was

signifi-cantly increased in most of the subsequent

reculture cycles The tallest shoot length

was not affected by explant orientation or

by reculture number This horizontal

recul-ture procedure was also applied to shoots

of clone HV derived from G+S explants

with similar results (figure 4).

The rooting performance of 431 and HV

clone shoots harvested from vertical and

horizontal culture systems was evaluated

Explant orientation during the first

multi-plication cycle of the explant did not affect

the rooting capacity of the shoots produced

in vertical (9 and 30% for clones HV and

431, respectively) and horizontal (9 and 26%

for clones HV and 431, respectively)

cul-tures However, reculture of the

horizon-tally placed explants significantly pos-itive effect (P < 0.01) on rooting

percent-age of shoots produced In 431 material, the

rooting frequency was increased nearly

two-fold from the first (26%) to the second

recul-ture (51%), and then leveled off The

high-est rooting percentage (62.8) as well as the

greatest root number (2.6) were achieved in shoots harvested from the sixth reculture,

being significantly higher than material derived from the first horizontal cycle Aver-age longest root length varied from 10 to

18.4 mm, but was not significantly affected

by shoot orientation or reculture number Similar trends were seen in clone HV, whose

rooting capacity is very poor In this clone,

rooting frequency and longest root length

were significantly increased from the first

(9% and 9 mm, respectively) to the fourth

horizontal reculture (28% and 18 mm,

respectively) Mean root number ranged

from 1.3 to 1.8, but no significant

differ-ences were found among different

treat-ments.

DISCUSSION

The data presented here show that suitable

reinvigoration methods have been

devel-oped to successfully micropropagate mature

chestnut trees from crown material These

results confirm that the in vitro response of

explants taken from the crown of

recalci-trant trees is very poor and, when juvenile

material is not available, reinvigoration

methods need to be applied in order to in

vitro clone and maintain selected genotypes

emphasized plantlets were obtained from untreated

mate-rial in two of five tested clones

In the present study, the BA spray

treat-ment did improve the in vitro reactivity of crown-derived explants, especially those derived from the very recalcitrant HV tree,

allowing them to be established in vitro On the contrary, explants exposed to short dura-tion pulses of BA were not reactivated

However, Read (1985) obtained microshoot

proliferation in several species, either by spraying the donor plant with cytokinins, including the same cytokinin in the medium,

or by soaking the explants in cytokinin prior

to establishment of cultures Pulse treatment

has also been shown as an alternative treat-ment to the incorporation of the cytokinin

into the medium for the induction of adven-titious buds in conifers (Von Arnold et al,

1988; Martinez Pulido et al, 1992) These

reports indicate that the concentration,

expo-sure time and mode of application are

impor-tant factors influencing the morphogenetic

response of explants Therefore, the observed different response of chestnut

explants to the BA applications (spray ver-sus pulse) may be due to the fact that the

BA spray was repeated six times and to the different concentration used in pulse and spray treatments The application frequency

of sprays has been reported to have an

important effect on the rejuvenation of Picea

abies, even more than the concentration

fac-tor (Bouriquet et al, 1985) The application

of BA in a water-ethanol solution to

Euca-lyptus ficifolia trees induced bud break from buds in the lignotuber as well as from latent buds in the upper trunk region (Mazalewsky

and Hackett, 1979) BA spraying of the

par-ent tree also improved the

micropropaga-tion of Platanus x Acerifolia (Donkers and

Evers, 1985), Pinus taeda (Timmis, 1985) and Pinus pinaster (Dumas, 1987) It is

sig-nificant that the initial BA-induced

rein-vigoration of chestnut explants was main-tained through out the multiplication stage

and affected the rooting capacity, which was

(1983) also reported that the foliar sprays

of BA applied to Pseudotsuga menziesii

were as effective as several successive

grafts If a gradual rejuvenation results from

conditions that stimulate multiplication of

juvenile cells (Greenwood, 1995), in this

case the BA, a more juvenile status could

then take place in the developed shoots

Therefore, cytokinins would act as a

reju-venating agent for mature trees (Franclet,

1981; Kelly, 1988).

Chestnut crown cultures from three

mature trees were successfully established in

vitro after a single grafting Grafting has

been reported previously as a suitable

method for inducing bud break and

subse-quent in vitro establishment of crown

mate-rial taken from one chestnut tree, but no

fur-ther data about multiplication and rooting

have been recorded (Ballester et al, 1990).

Rejuvenation of Sequoiadendron giganteum

has been reported by Monteuuis (1991) in

one of 300 grafted apex after only one

graft-ing and subsequent in vitro meristem

cul-ture As in other woody species

(Dooren-bos, 1965; Struve and Lineberg, 1988),

chestnut juvenile grafting causes a great

increase in both vigor and growth of shoots,

indicating an apparent partial rejuvenation.

It should be emphasized that our grafting

system fulfils the requirements for achieving

a successful reinvigoration: the high vigor of

the juvenile rootstock (Franclet, 1979), the

proximity of scion to the seedling root

(Chaperon, 1979; Paton, 1984) as well as

the small size of the scion (Stoutmyer and

Britt, 1961; Franclet, 1979)

A combination of both treatments,

graft-ing and BA spray, proved to be more

effec-tive for the in vitro establishment of

chest-nut crown material than either BA spray or

grafting alone, which agree with

prelimi-nary results in one clone of chestnut reported

by Ballester et al (1990) This treatment was

not successful for mature cultivars of walnut

trees (McGranaham et al 1987) The

reac-of explants grafted plus

BA sprayed material used in the present

work were similar to those of basal shoot

explants of the same clones, whereas the shoot multiplication rates and rooting capac-ities were not improved to the same extent as

reactivity (Sánchez and Vieitez, 1991) Therefore, the degree of reinvigoration depends on both the treatment being used and the characteristic being evaluated,

sup-porting the quantitative aspect of the

reju-venation as was suggested by Hackett (1985) These results show that both

treat-ments, grafting plus BA spray, and BA spray

alone, induce a partial rejuvenation from a

mature phase to a more physiologically

juve-nile state Moreover, the ex vitro behavior and other characteristics of this material have not been evaluated According to

Fontanier and Jonkers ( 1976), a true

onto-genetical rejuvenation occurs when an adult meristem produces plants completely

juve-nile

Because of the different morphogenetic

responses to in vivo treatments exhibited

by the clones HV (more recalcitrant) and

431 (more responsive), microshoots of both clones were selected for testing the effect

of recycling the same horizontal explant.

The morphogenetic response of clone 431 and HV material obtained by spraying with

BA and grafting plus BA spray treatments,

respectively, was highly enhanced by the horizontal reculture system It should be

emphasized that this method provided not

only the greatest proliferation rates, but also the best rooting frequencies, which were

similar for clone 431 (62.8%) or even higher

for clone HV (28.4%) than those reported

for microshoots originating from the base

of the same trees (51.4 and 19.4% for clones

431 and HV, respectively; Sánchez and Vieitez, 1991) Moreover, for clone 431, the

length of the tallest shoot was similar in the different reculture cycles even though the reculture period was reduced to 2 weeks after the second cycle Therefore, this method provides a more rapid and efficient